In the following description of the present specification, when nucleic acid bases are referred to, A, C, G, T and U it means adenine, cytosine, guanine, thymine and uracil, respectively. In addition, the term “oligonucleotide” optionally includes “polynucleotide”.
It has been known that, when a duplex RNA is introduced into a cell, an mRNA (messenger RNA) having a complementary base sequence to the RNA is decomposed and causes inactivation of the mRNA (RNA interference; abbreviated as “RNAi”). Such phenomenon has been considered to occur by the following mechanism.
That is, when a relatively long duplex RNA (dsRNA) is introduced into a cell, firstly the RNA is decomposed to 21 to 23 bases in size by an RNase III-like nuclease referred to as Dicer, and generates low molecular weight siRNA. Then, the siRNA binds to plural number of proteins to form a complex referred to as RISC (RNA induced silencing complex). The resulting complex recognizes and binds to an mRNA of the cell in which the same base sequence as the siRNA is contained, and then the mRNA is cleaved at the central part of siRNA. It has been considered that, in consequence, the relevant gene is inactivated.
The RNAi method (the RNA interference method) is a method to suppress an expression of a certain gene by introducing an artificially synthesized RNA into a cell utilizing this phenomenon, and has been used widely as a simple and potential method for inhibiting a gene function (see, for example, Fire, A. et. al., Nature, 1998, vol. 391, pp. 806-811; Svobada, P. et al., Development, 2000, vol. 127, pp. 4147-4156; Elbashir, S. M., Lendeckel, W. and Tuschl, T., Genes and Dev., 2001, vol. 15, pp. 188-200; Zamore, P. D. et al., Cell, 2000, vo. 101, pp. 25-33; Bernstein, E. et al., Nature, 2001, vol. 409, pp. 363-366).
In addition, compared to the antisense method described later, the RNAi method has advantages such as capability of suppressing the gene expression efficiently by a low concentration due to use of a mechanism possessed by living individual; low toxicity; high specificity to the target base sequence; and the simple experimental procedures. Also, the method has been used widely for knock-down of an endogenous gene in a cell. Further, an application of the method to gene therapy based on the specific decomposition of mRNA coding an abnormal gene has been expected.
On the other hand, however, the conventionally used low molecular weight RNA such as dsRNA and siRNA has a problem of difficulty in handling, because they are easily decomposed by an action of nucleic acid-hydrolyzing enzymes such as nucleases.
On the other hand, as an another method for controlling a gene expression, a technique named antisense method has also been known.
A base sequence of an mRNA for synthesis of a protein (for directing protein synthesis) is referred to as a sense sequence, and a complementary base sequence thereto is referred to as an antisense. In addition, a nucleic acid having a base sequence of an antisense is referred to as an antisense nucleic acid.
The antisense method is one of the methods for controlling an expression of a gene that are intended to suppress only a target gene specifically to the base sequence by administering the antisense nucleic acid being complementary to an mRNA transcripted from the certain target gene to the cell, and thereby forming a duplex between the mRNA of the cell and the antisense nucleic acid administered. Function of the target gene can be analyzed by evaluating inhibitory effect of the antisense nucleic acid on the target gene.
In addition, the antisense method can be applied to pharmaceutical field. For example, by binding an antisense nucleic acid to an mRNA, which is involved in synthetic process of a responsible protein factor for developing certain kind of disease, it can be expected that the function of the relevant gene will be prevented. Therefore, it is expected that, by using an antisense nucleic acid, treatment based on inhibition of action of causative gene for a disease can be performed. For example, Formivirsen, a medical drug for cytomegalovirus retinitis, which has been currently approved to ISIS Pharmaceuticals Inc. by FDA in the United States, is one example of this technology.
Properties required for the antisense nucleic acid include, for example, capability of forming a stable duplex with target RNA; base sequence-recognizing ability not to bind to a sequence containing mismatch base; resistance to nucleases; and cell membrane permeability. In addition, for using the antisense nucleic acid as a medical drug, recognition specificity to the base sequence, resistance to nucleases, antimetabolic property and intracellular mobility are of importance.
However, if naturally occurring type of oligonucleotide is used as an antisense nucleic acid, there are some problems that, for example, the required properties as described above including resistance to nucleases cannot be satisfied.
Therefore, up to the present, a number of trials on the modifications of nucleic acid have been conducted in order to obtain an oligonucleotide having properties of overcoming the drawback of the naturally occurring type of oligonucleotide, and also having properties being satisfied. This includes, for example, modification of base part of a nucleic acid (N. Haginoya et al., Bioconjugate Chem., 1997, vol. 8, pp. 271-280), modification of a ribose (M. Aoyagi et al., Bioorg. Med. Chem. Lett., 1996, vol. 6, pp. 1573-1576), alteration of a ribose ring itself (A. Kakefuda et al., Tetrahedron, 1996, vol. 52, pp. 2863-2876), modification to phosphodiester (M. Shimizu et al., 2006, vol. 71, pp. 4262-4269) and alteration of phosphodiester bond (A. Waldner et al., Bioorg. Med. Chem. Lett., 1994, vol. 4, pp. 405-408). By combining these techniques, diversification of the antisense nucleic acid, which can satisfy the properties as described above, can be expected. For example, S. M. Gryaznov et al. have synthesized 5′-phosphoroamidate type DNA (S. M. Gryaznov et al., Nucleic Acids Res., 1992, vol. 20, pp. 3403-3409). This type of DNA has resistance to nucleic acid degrading enzymes such as nucleases, but there remains a problem that binding affinity thereof to target mRNA is insufficient. To solve the problem of binding affinity to the target mRNA, S. Obika et al. have synthesized 5′-amino-2′,4′-bridged nucleic acid (BNA) containing a nucleotide which is constructed by introducing amino group at 5′-position of the sugar moiety and 2′- and 4′-positions are cyclized via an oxygen atom (S. Obika, et. al., Chem. Commun., 2003, pp. 2202-2203). In addition, Obika et al. also have synthesized 5′-amino-3′,5′-BNA in which 5′-amino group and 3′-position are bridged by methylene group (S. Obika, et al., Angew. Chem. Int. Ed., 2005, vol. 44, pp. 1945-1947). These BNAs have high binding affinity to target mRNA and resistance to nucleic acid degrading enzymes such as nucleases. However, there has not been any report on the protein suppression activity when these BNAs are introduced into siRNA.